Blood cells are produced from cells called hematopoietic stem cells through a process of proliferation and differentiation. The hematopoietic stem cells and blood cells at various differentiation stages are present in bone marrow. The discrimination and identification of the hematopoietic stem cells and blood cells in bone marrow and examining the change of the number or ratio thereof are scientifically and industrially significant for evaluating the toxicity of a drug including a medicine to the hematopoiesis in bone marrow.
In order to evaluate the toxicity to the hematopoiesis, it is necessary to discriminate and identify the hematopoietic stem cells and blood cells in bone marrow and clarify what actions occur in each lineage or differentiation stage. It is thought that there is a possibility of changing the number of erythroblasts and reticulocytes, for example in the case of drugs having toxicity to erythrocytes and varying the number of myelocytes (myeloblasts, promyelocytes, myelocytes, metamyelocytes or the like) or lymphocytes present in bone marrow in the case of drugs acting on leukocytes (lymphocytes, granulocytes or the like).
A method for preparing a smear, morphologically discriminating cells with human eyes by microscopic observation and identifying the cells has hitherto been mainly used for evaluating the toxicity to the hematopoiesis in bone marrow. The smear is prepared by thinly smearing a collected bone marrow sample on a slide glass and used by usually subjecting the smear to May-Grünwald and Giemsa staining or the like, then observing cell morphology under an optical microscope and determining how the ratio of respective cells varies. However, it is necessary to observe a large number of cells by a skilled person in charge of tests for evaluating the toxicity by the method and there are many subjects in the method in aspects of quality and quantity of date and time. Therefore, establishment of a method by which the problems can be overcome has been desired.
Flow cytometers have been utilized for collecting various qualitative or quantitative parameters in individual cells in fields of studies on cell biology, immunology and hematology. The flow cytometers are apparatuses designed to keep cells in a state of a suspension, passing the suspension through a fluid system at a high speed, instantaneously analyzing optical and electric signals obtained from the respective cells through a detecting part and studying and elucidating biological features of the respective cells. For example, the flow cytometers are utilized for measurement of the nucleic acid amount, enzyme activity, calcium influx, cell membrane potential, pH and the like (Malin-Berdel, J. and Valet, G. 1980. Cytometry 1:222-228; Phillip, R. et al. 1995. Cytometry 23: 322-329). It is known that the blood cells can be discriminated and identified by determining the quantity of expression of a cell surface antigen characteristic to each species of the cells by using the flow cytometers, for example, CD2 (Williams, A. F. et al. 1987.J. Exp. Med 165:368-380) and CD8 (Brideau, R. J. et al. 1980. Eur. J. Immunol. 10: 609-615) are known as specific for lymphocytes; CD11b is known as specific for granulocytes and myelocytes (Tamatani, T. F. et al. 1993. Eur. J. Immunol. 23: 2181-2188), CD18 (Tamatani, T. M. 1991. Eur. J. Immunol. 21: 627-633), CD45 (Sunderland, C. A. 1979. Eur. J. Immunol. 9: 155-159), CD45R.(Kroese, F. G. M. 1986, Adv. Exp. Med. Biol. 186: 81-89), CD90 (Campbell, D. G. 1981. Biochem. J. 195: 15-30) and the like are known as cell surface antigens recognized to be common to leukocytes. CD71 is known as a cell surface antigen correlating with the proliferating activity of cells (Jefferies, W. A. 1985. Immunolgy 54: 333-341).
The fact that rapid analysis can mechanically be carried out for a large number of cells with a small amount of a sample in a short time is cited as features of the method for using flow cytometers. Furthermore, the fact that the accuracy control important to assurance of data quality can be conducted and objective test data can be obtained is also cited as the features.
Johanna and Fridtjof describe the identification and isolation of respective progenitor cells of hematopoietic stem cells, myelocytes, lymphocytes and erythrocytes in normal adults or fetuses by using CD34, CD38, a cell adhesion molecule (L-selectin, CD18, CD33, CD44, CD48, CD49e, CD50 or CD52) and/or growth factor receptors (SCFR, GM-CSFR, IL-6R, gp130/IL-6R and IL-7R) as markers with flow cytometers (WO95/12813).
Terstappen describes the identification and discrimination of cell populations in peripheral blood or bone marrow in normal adults or fetuses using CD45 and CD71 as markers with flow cytometers [JP-A 6-27017 (hereunder, JP-A means “Japanese Unexamined Patent Publication”)].
Loken describes the discrimination and identification of bone marrow cells in normal adults by combining CD45 with various cell surface antigens (CD15, CD16, CD10, CD34, CD20, CD19, CD14, CD3 and CD11b) with flow cytometers (JP-A 1-161153).
In the above studies, it is shown that the method with the use of flow cytometers is useful when peripheral blood cells or bone marrow cells derived from human adults or fetuses; however, it is not disclosed that the method with the use of flow cytometers is similarly useful for experimental animals.
In general, it is not thought that facts confirmed in humans about what cell surface antigens are expressed in what blood cell in a process for proliferation and differentiation stages from hematopoietic stem cells can directly be extended to other animal species.
The above reports take the hematopoietic stem cell transplantation or the like into consideration, and the discrimination and identification of specific cell populations are considered as the main purpose of utilization. In order to utilize the method with the use of flow cytometers for evaluating the toxicity of drugs, it is, however, necessary to use cell surface antigen markers suitable for the purposes as an index.
It is heretofore not clear at all whether a suitable marker is present or whether the marker is actually useful even. if the marker is present. Namely, it is necessary to elucidate the correspondence of toxicological change of the number or the ratio of specific cell populations with the drugs possessing the hematopoietic toxicity to the change of the marker in question.
The above reports do not describe whether or not the identification and discrimination of cell populations can be carried out even for bone marrow cells derived from experimental animals by analyzing the cell surface antigens as markers with flow cytometers. Furthermore, the reports do not describe whether or not the markers in the reports are useful for evaluating the toxicity of a drug to hematopoiesis in bone marrow. Accordingly, the possibility of carrying out toxicity tests using experimental animals with flow cytometers could not be foreseen even on the assumption of the above reports.
Since the cell populations in bone marrow is heterogeneous as compared with peripheral blood, it is necessary to find out cell surface antigens to be an appropriate marker for discriminating and identifying the lineage of cells or differentiation stage with the use of flow cytometers. And then, it is necessary to elucidate that the utilization of the markers is useful for evaluating toxicity of a drug to hematopoiesis in bone marrow.
The flow cytometers are heretofore scarcely utilized in fields of toxicity. tests. Absence of investigation and substantiation of practicality and usefulness essential to utilization in toxicity tests, especially lack of data on expression patterns of cell surface antigens in hematopoietic stem cells in bone marrow of experimental animals (rodents such as rats) used in the toxicity tests or blood cells at various differentiation stages is cited as main reasons.
Examples of analyzing reticulocytes in peripheral blood with a flow cytometer and reporting that the evaluation of toxicity to erythrocytic hematopoiesis can be carried out by inventors of the present invention are cited as examples of utilizing the flow cytometer in toxicity tests (Miura, D. 1999. J. Toxicol. Sci. 24: 306). In the case of reticulocytes, measurement of the amount of intracellular RNA is a useful marker for identifying cells, and the investigation and substantiation as to establishment and usefulness of the method for testing using the flow cytometer can be made. However, there is no knowledge on what markers may be used for the method for testing toxicity to bone marrow hematopoiesis, especially leukocyte hematopoiesis with the flow cytometer.